Immune modulating inhibitory receptors

Abstract

The present invention relates to a method for screening for compounds which modulate the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell, in which a ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof with a test compound is contacted with a test compound; and the effect of the test compound on the expression and / or activity of the a ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof is determined. The invention also includes methods that relate to the inventive concept of using the ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell to test compounds. Immune cell wherein a gene encoding the ITIM and / or ITSM comprising modulatory immune receptor has been knocked-out is also disclosed.

Description

[0001] Title: Immune modulating inhibitory receptors

[0002] FIELD OF THE INVENTION

[0003]

[0001] This invention pertains in general to immune modulating receptors, (also referred to herein as modulatory immune receptors) and methods for screening for, identifying, and / or testing compounds / drugs that may modulate the expression and / or activity of such immune modulating receptors.

[0004]

[0002] In particular the invention relates to the identification of ITIM and / or ITSM comprising proteins that are expressed in various immune cells, and under different conditions.

[0005] BACKGROUND OF THE INVENTION

[0006]

[0003] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0007]

[0004] Blocking modulatory receptors like PD-1 and CTLA-4 has revolutionized cancer treatment in recent years. However, despite major successes in melanoma and lung cancer, the majority of cancer types are not responsive to these immunotherapies. As such, there is an ongoing need for the identification of novel modulatory receptors as drug targets, and compounds that can target these novel modulatory receptors and thereby modulate the expression and / or activity of such immune modulatory receptors.

[0005] In light of this, new products, compositions, methods and uses for in the treatment of conditions that involve such modulatory receptors would be highly desirable but are not yet readily available. In particular, there is a clear need in the art for reliable, efficient, and reproducible products, compositions, methods and uses that allow to be used in the treatment of such conditions. Accordingly, the technical problem underlying the present invention can been seen in the provision of such products, compositions, methods and uses for complying with any of the aforementioned needs, or at least providing the public with a useful choice. The technical problem is solved by the embodiments characterized in the claims and herein below. SUMMARY OF THE INVENTION

[0008]

[0006] As embodied and broadly described herein, the present invention is directed to the surprising identification of modulatory receptors that are expressed across a wide variety of immune cells, and show cell type-specific patterns in expression, for example depending of the cell state (resting, activated), and also in relation to the function of the subset or subtype or its environment. For example, the inventors surprisingly determined immune modulatory receptors are expressed differentially in CD4+ and CD8+ T cell subsets, including exhausted CD8+ T cells and CD4+ memory T cells, which may allow for subset-specific targeting.

[0009]

[0007] All this results from an analysis by means of a novel bioinformatics pipeline, which allowed to identify immune modulatory receptors as potential therapeutic targets.

[0010]

[0008] The invention refers in a first aspect to a method for screening for compounds which modulate the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell, more preferably wherein the ITIM and / or ITSM comprising modulatory immune receptor is i. a negative feedback modulatory immune receptor, preferably that is expressed in an activated immune cell; ii. a threshold modulatory immune receptor, preferably that is expressed on an immune cells in resting state; iii. a threshold-disinhibition modulatory immune receptor, preferably that is expressed on an immune cell in resting state and which expression is downregulated after activation of the immune cell; or iv. a threshold-negative feedback modulatory immune receptor, preferably expressed on an immune cell in resting state and which expression is upregulated after activation of the immune cell, comprising:

[0011] (a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof with a test compound; and (b) determining the effect of the test compound (which can also be a therapeutic element) on the expression and / or activity of the a ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof.

[0012]

[0009] Another aspect of the invention is a method for identifying a compound which modulates the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell, more preferably wherein the ITIM and / or ITSM comprising modulatory immune receptor is i. a negative feedback modulatory immune receptor, preferably that is expressed in an activated immune cell; ii. a threshold modulatory immune receptor, preferably that is expressed on an immune cells in resting state; iii. a threshold-disinhibition modulatory immune receptor, preferably that is expressed on an immune cell in resting state and which expression is downregulated after activation of the immune cell; or iv. a threshold-negative feedback modulatory immune receptor, preferably expressed on an immune cell in resting state and which expression is upregulated after activation of the immune cell, comprising:

[0013] (a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof with a test compound; and

[0014] (b) determining the effect of the test compound on the expression and / or activity of the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof to thereby identify a compound which modulates the activity of the polypeptide or biologically active portion thereof.

[0015]

[0010] Yet another aspect of the invention is a method for testing a drug for modulating the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell, more preferably wherein the ITIM and / or ITSM comprising modulatory immune receptor is i.a negative feedback modulatory immune receptor, preferably that is expressed in an activated immune cell; ii.a threshold modulatory immune receptor, preferably that is expressed on an immune cells in resting state; iii.a threshold-disinhibition modulatory immune receptor, preferably that is expressed on an immune cell in resting state and which expression is downregulated after activation of the immune cell; or iv.a threshold-negative feedback modulatory immune receptor, preferably expressed on an immune cell in resting state and which expression is upregulated after activation of the immune cell, comprising:

[0016] (a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof with the drug; and

[0017] (b) determining the effect of the drug on the expression and / or activity of the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof.

[0018]

[0011] The invention also relates in another aspect to a method for screening for compounds which modulate the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising:

[0019] (a) contacting the polypeptide or biologically active portion thereof with a test compound; and

[0020] (b) determining the effect of the test compound on the expression and / or activity of the polypeptide or biologically active portion thereof.

[0021]

[0012] Table 1 can be found in the Detailed Description section of this document.

[0022]

[0013] Another aspect of the invention is a method for identifying a compound which modulates the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising:

[0023] (a) contacting the polypeptide or biologically active portion thereof with a test compound; and

[0024] (b) determining the effect of the test compound on the expression and / or activity of the polypeptide or biologically active portion thereof to thereby identify a compound which modulates the activity of the polypeptide or biologically active portion thereof.

[0025]

[0014] Yet another aspect of the invention is a method for testing a drug for modulating the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising:

[0026] (a) contacting the polypeptide or biologically active portion thereof with the drug; and

[0027] (b) determining the effect of the drug on the expression and / or activity of the polypeptide or biologically active portion thereof. / nlp

[0028]

[0015] Another aspect of the invention is an immune cell wherein a gene encoding the ITIM and / or ITSM comprising modulatory immune receptor or a gene encoding the polypeptide encoded by a gene listed in Table 1 has been knocked-out.

[0029]

[0016] The invention also relates in another aspect to an expression vector comprising a nucleotide sequence encoding the ITIM and / or ITSM comprising modulatory immune receptor or encoding the polypeptide encoded by a gene listed in Table 1 , operably linked with a promoter.

[0030]

[0017] Another aspect of the invention is a host cell comprising the vector of the previous aspect, or recombinantly expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1.

[0018] The invention also relates in another aspect to a method of determining the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 in a cell comprising:

[0031] (a) providing the cell; and

[0032] (b) determining the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 in the cell, preferably wherein the cell is an immune cell.

[0033]

[0019] Another aspect of the invention is a method for identifying a binding partner to a ITIM and / or ITSM comprising modulatory immune receptor or a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising: (a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide or biologically active portion thereof, or a cell expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide, or biologically active portion thereof, with a test compound; and

[0034] (b) determining whether the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide or biologically active portion thereof binds to the test compound.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036]

[0020] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0037]

[0021] Figure 1 : A novel bioinformatics approach for the identification of predicted modulatory receptors. Schematic overview of the bioinformatics pipeline with the number of unique genes and corresponding proteins remaining at every step. All amino acid sequences corresponding to a protein-coding transcript were retrieved from Ensembl.

[0038]

[0022] Figure 2: Known and predicted single-spanning modulatory receptors are expressed in different cell types in the resting state and after activation. A. Novel and known receptors were classified into different functional categories based on changes, or lack thereof, in expression after stimulation. Threshold receptors were expressed at resting state, and did not change after activation (i.e. , change in expression < 0.5 Iog2 fold change). Threshold-negative feedback and threshold-disinhibition receptors were defined by > 0.5 Iog2 fold change up or downregulation, respectively, in expression after activation. Negative feedback receptors were absent in the resting state, but were expressed after activation. Categories indicated from left to right are shown from top to bottom in the figure. B. Upset plot showing the number of singlespanning receptors that are expressed uniquely by individual immune cell subsets , or shared between subsets as indicated by connected circles. Sixty-four genes are expressed in all cell types (not depicted).

[0039]

[0023] Figure 3: Single-spanning predicted modulatory receptors are expressed across a wide variety of tumor infiltrating T cell subsets. Number of modulatory receptor genes expressed by different CD4+ (A) and CD8+ (B) T cell subsets. Categories indicated from top to bottom are shown from left to right in the figure.

[0040]

[0024] Figure 4: Known and predicted multi-spanning modulatory receptors are expressed in different cell types in the resting state and after activation. A. Known and predicted multi-spanning receptors were classified into different functional categories based on changes, or lack thereof, in expression after stimulation. Threshold receptors were expressed at resting state, and did not change after activation (i.e. , change in expression < 0.5 Iog2 fold change). Threshold-negative feedback and threshold- disinhibition receptors were defined by > 0.5 Iog2 fold change up or downregulation, respectively, in expression after activation. Negative feedback receptors were absent in the resting state, but expressed after activation. Categories indicated from left to right are shown from top to bottom in the figure. B. Upset plot showing the number of multi-spanning receptors that are expressed uniquely by individual immune cell subsets , or shared between subsets as indicated by connected circles. Seventy-five genes are expressed in all cell types (not depicted). Cell subsets without uniquely expressed putative modulatory receptors, i.e., B cells and T cells, were excluded from the panel for clarity.

[0041]

[0025] Figure 5: Multi-spanning predicted modulatory receptors are expressed across a wide variety of tumor infiltrating T cell subsets. Number of modulatory receptor genes expressed by different CD4+ (A) and CD8+ (B) T cell subsets. Categories indicated from left to right are shown from left to right in the figure.

[0042] DESCRIPTION

[0043] Definitions

[0044]

[0026] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0045]

[0027] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0028] For purposes of the present invention, the following terms are defined below.

[0029] As used herein, the singular form terms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0046]

[0030] As used herein, “about” and “approximately", when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed invention. Unless otherwise clear from context, all numerical values provided herein include numerical values modified by the term “about.”

[0047]

[0031] As used herein, “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0048]

[0032] As used herein, "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... , etc. As used herein, the term "at most" a particular value means that particular value or less. For example, "at most 5" is understood to be the same as "5 or less" i.e., 5, 4, 3, ... .-10, -11 , etc.

[0049]

[0033] As used herein, “comprising” or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. It also encompasses the more limiting “to consist of”.

[0050]

[0034] As used herein, “conventional techniques” or “methods known to the skilled person” refer to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology and related fields are well-known to those of skill in the art and are discussed, in various handbooks and literature references.

[0051]

[0035] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and should not be construed as a limitation on the scope of the invention. The description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range including both integers and non-integers. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, 6 etc. This applies regardless of the breadth of the range.

[0052]

[0036] As used herein, “agonist”, refers to a compound or agent having the ability to initiate or enhance a biological function of a target protein or polypeptide, such as increasing the activity or expression of the target protein or polypeptide. Accordingly, "agonist" is defined in the context of the biological role of the target protein or polypeptide. While some agonists herein may specifically interact with (e.g., bind to) the target, compounds and / or agents that initiate or enhance a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target polypeptide is a member are in embodiments specifically included within this definition.

[0053]

[0037] As used herein, "antagonist" and / or "inhibitor" are used interchangeably, and they refer to a compound or agent having the ability to reduce or inhibit a biological function of a target protein or polypeptide, such as by reducing or inhibiting the activity or expression of the target protein or polypeptide. Accordingly, the terms "antagonist" and "inhibitor" are defined in the context of the biological role of the target protein or polypeptide. While some antagonists herein may specifically interact with (e.g., bind to) the target, compounds that inhibit a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target protein or polypeptide are in embodiment also included within this definition.

[0054]

[0038] As used herein a “compound for screening”, “test compound”, “candidate agent” or “agent,” refers to a molecule that may be screened for, or be identified as, modulating activity of a target activity (e.g. a compound directly or indirectly interacts with a ITIM and / or ITSM comprising immune receptor expressed in an immune cell as identified and disclosed herein). Such agent may, for example, be an inhibitor or enhancer of the activity or expression and may find use in a variety of applications, including as therapeutic agents, as agricultural chemicals, and so on. The screening methods will typically be assays which provide for qualitative / quantitative measurements of the activity in the presence of a particular candidate agent. For example, the assay could be an assay which measures the immune cell release of certain cytokines, the expression of certain markers or the immune cell viability. The screening method may be an in vitro or in vivo format, and both formats are readily developed by those of skill in the art according to conventional techniques. (Candidate) agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts or purified compounds are available or may be produced. Additionally, natural or synthetically produced libraries and compounds can be prepared using conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs or derivates. (Candidate) agents may also be biomolecules including peptides, antibodies, ligands, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof, preferably antibodies, ligands or antigen binding molecule (preferably the antigen being comprised in the immune modulating receptor according to the invention). In a preferred embodiment antibodies Within the context of the current invention, the agent used in the various methods of the invention may include cells such a modulates cells, including living cells).

[0055]

[0039] Using the above screening methods, a variety of different therapeutic agents may be identified. For example, such agents may target a ITIM and / or ITSM comprising immune receptor expressed in an immune cell as identified and disclosed herein. Such agents may be inhibitors or promoters of the targeted activity, where inhibitors are those agents that result in at least a reduction of activity as compared to a control and enhancers result in at least an increase in activity as compared to a control. Such agents may be find use in a variety of (therapeutic) applications.

[0056]

[0040] As used herein, the term “immune cell” encompasses any cell involved in the innate or adaptive immune response and includes one or more of macrophages, neutrophils, dendritic cells, lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, granulocytes, T lymphocytes (including the killer T cell also known as CD8+ T cells, the helper T cell also known as CD4+ T cells, regulatory T cells, and ‘unconventional’ T cells exemplified by yb T cells and NKT cells), and B lymphocytes.

[0041] As used herein, the term “modulatory immune receptor” or “immune modulatory receptor” or “modulatory receptor” or “immune modulating receptor” broadly refers to an immune cell receptor that may regulate (positively or negatively) immune cell function to (enhance or reduce) modulate immune reactions, for example, modulate autoimmune reactions and / or immunopathology. Only by way of example, and without limiting the invention disclosed herein, the most well-described modulatory receptor mechanism of action is the local and transient intracellular attenuation of positive signals from activating receptors, including antigen-receptors and co-stimulatory receptors. Most of the known modulatory receptors attenuate T-cell receptors (TOR) or B-cell receptors (BCR) signalling events by targeting these directly or their downstream signalling molecules. Modulatory receptors cause a broad, quantitative modulation, for example reduction, in activation-induced signal transduction and downstream gene expression. In some embodiments the modulatory immune receptor is a inhibitory immune receptor. In some embodiments the modulatory immune receptor is a stimulatory immune receptor.

[0057]

[0042] As used herein, the term “immunoreceptor tyrosine-based modulatory motif (ITIM)” refers to a conserved sequence of amino acids that is found in the cytoplasmic tails of many modulatory receptors of the immune system. The ITIM motif generally comprises a serine residue (S), a isoleucine residue (I), a valine residue (V) or a leucine residue (L), separated by any other amino acid residue (x) from a tyrosine residue (Y), separated by any two other amino acids from a isoleucine residue (I), valine residue (V) or leucine residue (L). The consensus signature is S / l / V / LxYxxl / V / L. In vivo, ITIM-possessing modulatory receptors interact with their ligand, causing the ITIM motif to become phosphorylated by enzymes of the Src kinases, allowing them to recruit other enzymes.

[0058]

[0043] As used herein, the term “immunoreceptor tyrosine-based switch motif (ITSM)” refers to a conserved sequence of amino acids that is found in the cytoplasmic tails of many modulatory receptors of the immune system. The ITSM motif generally comprises a threonine residue (T), separated by any other amino acid residue from a tyrosine residue (Y), separated by any other two amino acids from a valine residue (V) or an isoleucine residue (I). The consensus signature is TxYxxV / l. In vivo, ITSM.

[0059]

[0044] As used herein, a “negative feedback modulatory immune receptor” is an modulatory immune receptor, which is induced by cellular activation to ensure a timely resolution of the response and limit excessive immune response.

[0060]

[0045] As used herein, a “threshold modulatory immune receptor”, is an modulatory immune receptor, which is expressed on immune cells in the resting state and control the threshold for immune cell activation to provide context and prevent unnecessary or futile responses.

[0061]

[0046] As used herein, a “threshold-disinhibition modulatory immune receptor” is a threshold modulatory immune receptor, which preferably it is expressed on an immune cell in resting state and which expression is downregulated after activation of the immune cell.

[0062]

[0047] As used herein, a “threshold-negative feedback modulatory immune receptor”, is a threshold modulatory immune receptor, which expression is further induced upon cellular activation.

[0063]

[0048] As used herein, it is be understood that a cell, preferably an immune cell is in a resting state when it is quiescent and does, for example, not proliferate to perform their function. On the contrary, when a cell, in particular any immune cell is activated, this means that by stimulatory and co-stimulatory signals from outside or inside the cell may, for example, switch to the proliferating state to perform their function. Other examples of activation may include, for example, cytokine production, cytotoxicity and / or degranulation. The skilled person is well aware of such and other functions of activated immune cells.

[0064]

[0049] As used herein, a “modulator” refers to a compound that is either an agonist or an antagonist. As used herein, a “modulator” refers to a compound that alters the activity of a target activity, for example the activity of a target protein. The modulator may be an inhibitor (antagonist) or an enhancer (agonist). The modulator may alter the activity by modulation of, for example, the enzymatic activity of a target protein, by modulation the interaction of the target protein with a further factor, such as a further protein, by modulation of the activity of a regulator of the target protein, and / or by modulating expression of the target protein. In one example, the modulator is a ligand.

[0050] As used herein, the terms “in vivo”, “ex vivo”, or “in vitro” take the meaning widely accepted. In vitro (Latin: in glass) studies in experimental biology are those that are conducted using components of an organism that have been isolated from their usual biological surroundings in order to permit a more detailed or more convenient analysis than can be done with whole organisms. In contrast, in vivo work is that which is conducted with living organisms in their normal, intact state, while ex vivo studies are conducted on functional organs that have been removed from the intact organism. Ex vivo (Latin: "out of the living") means that which takes place outside an organism. In science, ex vivo refers to experimentation or measurements done in or on tissue in an artificial environment outside the organism with the minimum alteration of natural conditions. Ex vivo conditions allow experimentation under more controlled conditions than is possible in in vivo experiments (in the intact organism), at the expense of altering the "natural" environment.

[0065]

[0051] As used herein, the term “expression vector” refers to a polynucleotide sequence, also known as plasmid or virus genome that comprises the sequence of a polynucleotide that is desired to be transcribed and translated in a cell. An expression vector, is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. The vector is engineered and contains regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The promoter and enhancers are operably linked to the polynucleotide that wants to be expressed in the cell or by means of the cell.

[0066]

[0052] As used herein the term “nucleic acid” or “polynucleotide” refers to any polymers or oligomers of (contiguous) nucleotides. The nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in singlestranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. The present invention also contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced.

[0067]

[0053] As used herein , “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleotide sequence. For instance, a promoter, or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked may mean that the DNA sequences being linked are contiguous.

[0068]

[0054] As used herein, “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin. A “fragment” or “portion” of a protein may thus still be referred to as a “protein.” A protein as defined herein and as used in any method as defined herein may be an isolated protein. An “isolated protein” is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

[0069]

[0055] As used herein, “sequence” or “nucleotide sequence” refers to the order of nucleotides of, or within a nucleic acid. In other words, any order of nucleotides in a nucleic acid may be referred to as a sequence or nucleotide sequence. The term “amino acid sequence” refers to the order of amino acids or, or within a polypeptide (or protein). In other words, any order of amino acids in a polypeptide may be referred to as a sequence of amino acid sequence.

[0070] Detailed description

[0071]

[0056] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0072]

[0057] It is contemplated that any method, use, or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0073]

[0058] Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions, and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.

[0074]

[0059] As embodied and broadly described herein, the present invention is directed to the surprising identification of modulatory receptors that are expressed across a wide variety of immune cells, display cell type-specific patterns in expression depending of the cell state (resting, activated), and in relation to function of the subset or subtype of cells or its environment. For example, the inventors surprisingly identified immune modulatory receptors are expressed differentially in CD4+ and CD8+ T cell subsets, including exhausted CD8+ T cells and CD4+ memory T cells, which allows for subsetspecific targeting.

[0075]

[0060] As previously indicated, the invention refers in a first aspect to a method for screening for compounds which modulate the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell, more preferably wherein the ITIM and / or ITSM comprising modulatory immune receptor is i. a negative feedback modulatory immune receptor, preferably that is expressed in an activated immune cell; ii. a threshold modulatory immune receptor, preferably that is expressed on an immune cells in resting state; iii. a threshold-disinhibition modulatory immune receptor, preferably that is expressed on an immune cell in resting state and which expression is downregulated after activation of the immune cell; or iv. a threshold-negative feedback modulatory immune receptor, preferably expressed on an immune cell in resting state and which expression is upregulated after activation of the immune cell, comprising: (a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof with a test compound; and

[0076] (b) determining the effect of the test compound on the expression and / or activity of the a ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof.

[0077]

[0061] In another aspect, the invention relates to a method for identifying a compound which modulates the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell, more preferably wherein the ITIM and / or ITSM comprising modulatory immune receptor is i. a negative feedback modulatory immune receptor, preferably that is expressed in an activated immune cell; ii. a threshold modulatory immune receptor, preferably that is expressed on an immune cells in resting state; iii. a threshold-disinhibition modulatory immune receptor, preferably that is expressed on an immune cell in resting state and which expression is downregulated after activation of the immune cell; or iv. a threshold-negative feedback modulatory immune receptor, preferably expressed on an immune cell in resting state and which expression is upregulated after activation of the immune cell, comprising:

[0078] (a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof with a test compound; and

[0079] (b) determining the effect of the test compound on the expression and / or activity of the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof to thereby identify a compound which modulates the activity of the polypeptide or biologically active portion thereof.

[0080]

[0062] The skilled person will know the experimental and assays commonly used to determine the activity of a receptor, in particular of an immune receptor. For example, the assay could be an assay which measures the immune cell release of certain cytokines, which are known to be released in an activated state; the expression of certain markers; or the immune cell viability.

[0063] Yet in another aspect, the invention relates to a method for testing a drug for modulating the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell, more preferably wherein the ITIM and / or ITSM comprising modulatory immune receptor is i.a negative feedback modulatory immune receptor, preferably that is expressed in an activated immune cell; ii.a threshold modulatory immune receptor, preferably that is expressed on an immune cells in resting state; iii.a threshold-disinhibition modulatory immune receptor, preferably that is expressed on an immune cell in resting state and which expression is downregulated after activation of the immune cell; or iv.a threshold-negative feedback modulatory immune receptor, preferably expressed on an immune cell in resting state and which expression is upregulated after activation of the immune cell, comprising:

[0081] (a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof with the drug; and

[0082] (b) determining the effect of the drug on the expression and / or activity of the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof.

[0083]

[0064] In a particular embodiment of any one of the previous aspects, the ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell is encoded by a gene listed in Table 1 .

[0084]

[0065] The invention also relates in another aspect to a method for screening for compounds which modulate the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising:

[0085] (a) contacting the polypeptide or biologically active portion thereof with a test compound; and

[0086] (b) determining the effect of the test compound on the expression and / or activity of the polypeptide or biologically active portion thereof.

[0066] Another aspect of the invention is a method for identifying a compound which modulates the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising:

[0087] (a) contacting the polypeptide or biologically active portion thereof with a test compound; and

[0088] (b) determining the effect of the test compound on the expression and / or activity of the polypeptide or biologically active portion thereof to thereby identify a compound which modulates the activity of the polypeptide or biologically active portion thereof.

[0089]

[0067] Yet another aspect of the invention is a method for testing a drug for modulating the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising:

[0090] (a) contacting the polypeptide or biologically active portion thereof with the drug; and

[0091] (b) determining the effect of the drug on the expression and / or activity of the polypeptide or biologically active portion thereof.

[0092]

[0068] In a particular embodiment of any of the methods of any of the aspects in this detailed description, the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide has the ability to modulate immunomodulatory function, modulate cytokine expression, modulate immune cell activation, modulate migration ability, modulate killing ability, and / or modulate cellular proliferation.

[0093]

[0069] In another particular embodiment of any of the methods of any of the aspects in this detailed description, the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide is expressed in a cell, preferably an immune cell.

[0094]

[0070] In yet another particular embodiment of any of the methods of the aspects of the invention, the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide is expressed in a cell, preferably an immune cells, and wherein the contacting with the test compound or drug is performed in the presence of a target of the cell, preferably immune cell.

[0095]

[0071] In yet another particular embodiment of any of the methods of the aspects of the invention, the immune cell is a neutrophil, a monocyte, a myeloid cell, a lymphoid cell, a B cell, a T cell, a NK cell, a helper T cell, a follicular helper T cell, an effector memory T cell, a regulatory T cell, a macrophage.

[0072] In yet another particular embodiment of any of the methods of the aspects of the invention, the T cell is a CD4+ T-cell, a CD8+ T cell, a tumor infiltrating T cell, an exhausted CD8+ T cell, a CD4+ memory T cell.

[0096]

[0073] In yet another particular embodiment of any of the methods of the aspects of the invention, determining the effect of the test compound or the drug on activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide, comprises determining an effect of the test compound or the drug on the immune response of the cell, preferably the immune cell expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide.

[0097]

[0074] In a more particular embodiment, the effect of the test compound or the drug on the immune response of the cell is modulating immunomodulatory function, modulating cytokine expression, modulating immune cell activation, modulating migration ability, modulating killing ability, and / or modulating cellular proliferation.

[0098]

[0075] In another particular embodiment of any of the methods of the aspects of the invention, the method comprises contacting the test compound or the drug with a cell expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide and contacting the test compound or the drug with an identical cell but not expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide or with an identical cell with reduced expression of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide or the polypeptide, and comparing the effect of the test compound or the drug on the expression and / or activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide.

[0099]

[0076] In another particular embodiment of any of the methods of the aspects of the invention, the cell is an immune cell and wherein the immune cell is activated prior to, during and / or subsequent to contacting the test compound or the drug with the cell.

[0100]

[0077] In another particular embodiment of any of the methods of the aspects of the invention, a further compound is contacted with the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide prior to, during and / or subsequent to contacting the test compound or the drug with the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide, preferably wherein the further compound is a known modulatory immune receptor modulator.

[0101]

[0078] In another particular embodiment of any of the methods of the aspects of the invention, the cell is a cell obtained from a subject, preferably wherein the cell is obtained from a subject suffering from cancer or suffering from an immune disease or suffering from inflammation. In one example, the subject are patients who enter or suspected to enter states of immune disease of inflammation due to iatrogenic intervention (e.g. checkpoint inhibitor treatment, allogenic stem cell transplantation .

[0079] More particular embodiments relate to cells from a subject suffering from a cancer selected from bladder cancer, brain cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, multiple myeloma, pancreatic cancer, prostate cancer, sarcoma, skin cancer, stomach cancer, endometrial cancer, among others.

[0102]

[0080] In another particular embodiment, the cell is obtained from a subject suffering from cancer or suffering from an immune disease selected from an autoimmune disease, for example, Multiple sclerosis, Myasthenia gravis, Pernicious anemia, Reactive arthritis, Rheumatoid arthritis, Sjogren syndrome, Systemic lupus erythematosus, Systemic sclerosis, Type I diabetes. Other examples of immune disease include asthma, eczema, allergic rhinitis, severe combined immunodeficiency (SCID) and temporary acquired immune deficiencies.

[0103]

[0081] In some embodiments, the cells obtained from a subject are cells obtained from the tissue, organ or area that is affected by such conditions as indicated herein. In some embodiments, the cells are obtained from a tissue, organ or area that is not affected by such conditions as indicated herein. In some embodiments, the cells are obtained from blood obtained from the subject.

[0104]

[0082] In another particular embodiment of any of the methods of the aspects of the invention, optionally in combination with any of their embodiments, the step of contacting occurs in vivo, ex vivo, or in vitro.

[0105]

[0083] As indicated, another aspect of the invention relates to an immune cell wherein a gene encoding the ITIM and / or ITSM comprising modulatory immune receptor or a gene encoding the polypeptide encoded by a genes listed in Table 1 has been knocked-out.

[0106]

[0084] The invention also relates in another aspect to an expression vector comprising a nucleotide sequence encoding the ITIM and / or ITSM comprising modulatory immune receptor or encoding the polypeptide encoded by a gene listed in Table 1 , operably linked with a promoter. Such expression vector and how to obtain them in the context of the current invention are well-known to the skilled person.

[0107]

[0085] Another aspect of the invention is a host cell comprising the vector of the previous aspect, or recombinantly expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1.

[0108]

[0086] The invention also relates in another aspect to a method of determining the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 in a cell comprising:

[0109] (a) providing the cell; and

[0110] (b) determining the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 in the cell, preferably wherein the cell is an immune cell.

[0111]

[0087] In a particular embodiment of the method of determining the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 , the cell, prior to determining the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 , is contacted with a test compound or drug as defined or identified in any of the aspects and their embodiments and including: a method for screening for compounds which modulate the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell; a method for identifying a compound which modulates the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell; a method for testing a drug for modulating the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell; a method for screening for compounds which modulate the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof; a method for identifying a compound which modulates the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof; and a method for testing a drug for modulating the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof.

[0112]

[0088] As will be understood by the skilled person, any method of screening, identifying or testing as disclosed herein may be performed in an immune cell expressing the, or more than one, modulatory immune receptor, or in a non-immune cell expressing the, or more than one, modulatory immune receptor, for example, adapted to express the, or more than one, modulatory immune receptor as disclosed herein. Preferably, the cell is an immune cell, more preferably, the cell is an immune cell naturally (in vivo) expressing the, or more than one, modulatory immune receptor.

[0089] In another particular embodiment of the method of determining the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 in a cell, the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 is, preferably independently, determined in more than one cell type, preferably wherein the more than one cell type is obtained from a subject, preferably from a subject suffering from cancer or suffering from an immune disease or suffering from inflammation.

[0113]

[0090] Finally, the invention also relates in another aspect to a method for identifying a binding partner to a ITIM and / or ITSM comprising modulatory immune receptor or a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising:

[0114] (a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide or biologically active portion thereof, or a cell expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide, or biologically active portion thereof, with a test compound; and

[0115] (b) determining whether the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide or biologically active portion thereof binds to the test compound.

[0116]

[0091] Embodiments of the previous aspects in this descriptions relating to type of cells also apply to any other aspect according to the invention.

[0117]

[0092] As already described herein, the polypeptide according to the invention or the modulatory immune receptor according to the invention for use in the various aspects according to the invention as disclosed herein is in a preferred embodiment encoded by a gene listed in Table 1 . In a more preferred embodiment, the polypeptide according to the invention / the modulatory immune receptor according to the invention / the gene according to the invention is selected from Table 2a, 2b, 2c, 2d, 3a, 3a”, 3b, 3b” or 4. In an even more preferred embodiment, the polypeptide according to the invention / the modulatory immune receptor according to the invention / the gene according to the invention is at least expressed in one or more, preferably one of the immune cells indicated in Table 2a, 2b, 2c, 2d, 3a, 3a”, 3b, 3b” or 4. For example, in one embodiment, the polypeptide according to the invention / the modulatory immune receptor according to the invention is one that is expressed in CD4 T cells, or one that is expressed in CD8 T cells, or expressed in B cells, or expressed in NK cells, or expressed in monocytes or expressed in neutrophils, either under resting or under stimulated conditions (see, for example Table 2, for example 2a, 2b), or is one that is expressed in a particular subset of CD4 T cells and / or CD8 cell, such as Th17 CD4 T cells or resident memory CD8 T cells, as indicated in Table 2c and / or 2d. In another example, the polypeptide according to the invention / the modulatory immune receptor according to the invention is one that is expressed in any on of the immune cells and is categorized to at least one of the categories as indicated in Table 3, in particular 3a, 3a”, 3b, and / or 3b”. In yet another preferred embodiment, the polypeptide according to the invention / the modulatory immune receptor according to the invention is one that is identified in Table 4. As will be understood by the skilled person, in a preferred embodiment, the polypeptide according to the invention / the modulatory immune receptor according to the invention is expressed in the corresponding immune cells, and / or under the corresponding conditions as indicated in any one of Table 2a, 2b, 2c, 2d, 3a, 3a”, 3b, 3b” or 4. In some embodiment, there is provided for a combination of the preferred embodiment described above.

[0118]

[0093] In yet another embodiment there is provided for the methods of the invention disclosed herein, wherein the method is for screening / identifying / testing / validating a compound for modulating the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell as defined in any of table 2a, 2b, 2c, 2d, 3a, 3a”, 3b, 3b” or 4.

[0119]

[0094] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[0120]

[0095] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[0121]

[0096] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[0122]

[0097] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments, and preferences for all aspect separately.

[0123]

[0098] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art.

[0124]

[0099] Table 1. All 390 modulatory receptor gene symbols, and corresponding matched Ensemble peptide IDs (release 105, December 2021 , in https: / / w w.ensembl.org / index.html).

[0125]

[0126]

[0127]

[0128] EXAMPLES

[0129] Example

[0130] Identification of ITIM / ITSM-bearing immune modulatory receptors

[0100] All the materials and methods for the performance of the examples are listed at the end of this section.

[0131] RESULTS

[0132] Putative ITIM / ITSM-bearing immune modulatory receptors can be found in the human genome

[0133]

[0101] To identify putative novel modulatory receptors, we first retrieved the protein sequences of all 96,457 protein-coding transcripts, corresponding to 19,353 individual human genes, annotated in the Ensembl database (release 105, December 2021)

[0013] , We retrieved all deposited isoforms, as certain proteins might only contain an ITIM in a specific isoform. We then screened the entire protein sequence for the presence of an ITIM or ITSM, based on the consensus sequence (V| L| I |S|T)xYxx(l | L|V), resulting in 40,063 protein sequences from the initial 96,457. We next used the TOPCONS server, which is based on a consensus of several prediction tools

[0014] , to determine the membrane topology of all proteins with an ITIM / ITSM motif. TOPCONS predicted 12,576 out of the 40,056 input proteins to be integral membrane proteins, of which 7,445 contained multiple transmembrane domains and the remaining 5,131 are singlepass membrane proteins.

[0134]

[0102] We next filtered proteins for the presence of the ITIM or ITSM sequence in an intracellular domain of the protein based on the predicted topology. This yielded with 4,613 protein sequences bearing one or more intracellular ITIMs or ITSMs, which correspond to 1 ,562 genes (Figure 1). To assess how well our pipeline reflects previously documented modulatory receptors, we confirmed that all the 52 proteins known for their ITIM-mediated immune modulatory effects, such as PD-1 or leukocyte- associated immunoglobulin-like receptor 1 (LAIR-1) (Table A)[7], were recalled successfully, with the exception of MPIG6B, encoding for the inhibitory receptor G6B on platelets and megakaryocytes

[0015] , due to an incorrect prediction of its transmembrane domain.

[0135] Table A. Genes encoding previously described ITIM-bearing inhibitory receptors

[0136] BTLA CLEC4A KIR3DL2 NCR2 SIGLEC11

[0137] CD22 FCGR2B KIR3DL3 PDCD1 SIRPA

[0138] CD244 FCRL2 KLRC1 PECAM1 SIT1

[0139] CD300A FCRL3 KLRG1 PILRA SLAM F6

[0140] CD300LF FCRL4 LAIR1 PVR TIGIT

[0141] CD33 FCRL5 LILRB1 SIGLEC5 TREM L1

[0142] CD5 KIR2DL1 LILRB2 SIGLEC6 VSIG4

[0143] CD72 KIR2DL2 LILRB3 SIGLEC7 VSTM1

[0144] CEACAM1 KIR2DL3 LILRB4 SIGLEC8

[0145] CLEC12A KIR2DL4 LILRB5 SIGLEC9

[0146] CLEC12B KIR3DL1 MPIG6B SIGLEC10

[0147]

[0103] The composition of an ITIM / ITSM sequence is highly variable, as only 3 out of 6 amino acids positions are limited to specific amino acids. As a result of this, large proteins or proteins containing many tyrosines and hydrophobic residues are more likely to contain an ITIM / ITSM by chance (Data not shown). We therefore determined the likelihood of encountering an ITIM / ITSM by chance in the intracellular region of the putative targets. To this end, we randomly permutated the amino acid sequences of the intracellular domains by shuffling their order using the Fisher-Yates algorithm. We did this 10,000 times for each receptor and assessed how often the permutated sequence contained more or an equal amount of ITIM / ITSMs compared to the original sequence. For 41 out of 51 known receptors, these odds were low (< 5%). However, for 10 out of 51 , we observed ITIM / ITSM sequences in the permutated sequence up to -25% of the time. Based on these findings, we decided to exclude novel targets when they were above this threshold of 25%. After this statistical filtering, we were left with 837 modulatory receptor genes. As expected, the average length of the intracellular domains of the excluded proteins was much higher than that of the remaining targets.

[0148] AlphaFold structure predictions can ass / st in identifying likely functional ITIM / ITSMs

[0149]

[0104] Protein interaction motifs are most commonly found in intrinsically disordered regions within proteins

[0016] , as structured regions are less accessible to binding with interacting partners. We therefore hypothesized that ITIM / ITSM sequences locate in disordered regions to allow for interaction with phosphatases and kinases. We therefore leveraged AlphaFold to infer three-dimensional structure predictions of the remaining modulatory receptor candidates to determine where the ITIM / ITSM sequences were located in three dimensional space

[0017] ,

[0150]

[0105] For 832 of the 837 remaining modulatory receptor genes, we were able to retrieve the predicted structures using AlphaFold. For every residue in the three- dimensional model, AlphaFold defines a confidence score (pLDDT). Low scores indicate reduced confidence, and regions with a low score are more likely to be intrinsically disordered

[0018] . We determined the average pLDDT of all six residues encompassing the ITIM / ITSM sequence, what resulted in an overall confidence score for each intracellular ITIM / ITSM in all targets.

[0151]

[0106] We found that 99 out of 101 ITIM / ITSMs of the 51 known receptors had low confidence scores, i.e. , less than 80 pLDDT, with an average confidence score of 49.3 pLDDT. 64 of the ITIMs even had pLDDT values below 50, which means they are particularly likely to be intrinsically disordered

[0018] . This result confirmed the hypothesis that ITIM / ITSMs are likely to be located in disordered regions in the protein and indicates that the AlphaFold confidence score can be a valuable asset to determine the potential functionality of an ITIM sequence in predicted targets.

[0152]

[0107] Based on the known receptors, we determined a threshold of 80 pLDDT and were left with 390 out of 832 putative ITIM-bearing modulatory receptor genes likely to contain a functional ITIM / ITSM. Among these genes, 54.6% consisted of multispanning proteins, while the remaining 177 single transmembrane domain targets consisted of 145 (37.2%) type I and 32 (8.2%) type II receptors. In some cases, a single gene was predicted to have both single-spanning and multi-spanning isoforms. Most putative modulatory receptors (275) were based on the canonical transcript, as annotated by Ensembl, while 115 were identified as a non-canonical isoform of the gene.

[0153] Putative modulatory receptors are expressed across immune cell subsets

[0154]

[0108] To better characterize these receptors, we studied the expression patterns of the retrieved known and putative modulatory receptor genes in various immune cells. We collected publicly available bulk RNA sequencing data of various immune cell subsets, both at resting state and after in vitro stimulation[19,20] (Table B). We included all putative modulatory receptors, including those that were identified in a non-canonical transcript of the gene. For this analysis, we considered a receptor as expressed when the expression was above the median overall gene expression in the sample, in either resting or activated state. From our set of modulatory receptor genes, 2 / 51 known and 41 / 390 predicted genes were not detected in these datasets.

[0155] Table B. Number of samples for different resting and activated immune cell subsets cell type subsets resting activated stimulation duration

[0156] Neutrophils 3 10 TSLP / S. 4h / 24h aureus

[0157] Monocytes 4 8 LPS 24h

[0158] NK cells 6 6 IL-2 24h

[0159] B cells naive, memory, 13 10 anti-IgG / M + 24h plasmablast IL-4 naive, effector, memory, dynabeads +

[0160] CD4 T cells 37 40 24h regulatory, Tfh IL-2

[0161] CD8 T cells naive, effector, memory 15 16 dynabeads + 24h

[0162] IL-2

[0109] Almost all functionally characterized immune modulatory receptors are type I transmembrane proteins, while only six are type II single-spanners. So far, no multispanning receptors have been described to have an modulatory function in the immune system. Also, many genes encoding multi-spanning proteins only showed very limited expression across immune cell subsets in the datasets we used for our analysis. Therefore, we analyzed the multi-spanning targets (Figure 4) separately from the single-spanning novel targets (Figure 2).

[0163]

[0110] We investigated the expression of 215 single-spanning receptor genes in these immune cell subsets and found that neutrophils (137 genes) and monocytes (120 genes) expressed the highest number of modulatory receptor genes. Lymphoid cells expressed fewer modulatory receptor genes compared to myeloid cells, with B and NK cells expressing 111 genes, followed by CD8+ (106 genes) and CD4+ (99 genes) T cells (Figure 2A). The relative contribution of known and novel targets in each subset varied between different immune cells. We found more novel targets being expressed in CD4+ (86 / 99 genes, 86.9%) and CD8+ T cells (88 / 106 genes, 84.3%), compared to neutrophils (98 / 137 genes, 71.5%). Neutrophils and monocytes uniquely expressed 9 and 3 modulatory receptor genes respectively, but the vast majority were shared between two or more immune cell subsets (Figure 2B).

[0164]

[0111] Compared to the single-spanners, we found slightly fewer multi-spanning modulatory receptor genes expressed in the different immune cell subsets. Out of 197 predicted genes, neutrophils expressed the most multi-spanning modulatory receptor genes (118 genes), while the other subsets expressed a similar number: Monocytes expressed 100 modulatory receptor genes, followed by B cells (95 genes), CD4+ (90 genes) and CD8+ (90 genes) T cells, and NK cells (89 genes) (Figure 4B). Neutrophils (14 genes) expressed the most unique set of multi-spanning modulatory receptors, while most other genes were shared between all subsets (Figure 4B).

[0165] Known and novel immune modulatory receptors of different functional categories can be identified in immune cells

[0166]

[0112] We next assigned the putative modulatory receptor into the previously described functional categories by comparing the expression in the resting state versus after activation in the available data from different cell types[7] (Table C). We defined negative feedback receptors as below median expression in the resting state, but expressed after activation. Threshold receptors were defined as being expressed in the resting state and having less than 0.5 Iog2 fold change after activation, while threshold-disinhibition and threshold-negative feedback receptors were defined by more than 0.5 Iog2 fold decrease or increase in expression upon activation, respectively. Using this functional classification system, we found that all immune cell subsets, except B cells, expressed receptors of all four categories, although all subsets primarily expressed threshold receptors.

[0167] Table C. Number of single-spanning receptors in different functional categories for each immune cell subset thresholdnot negative thresholdcell type negative threshold expressed feedback disinhibition feedback

[0168] Neutrophils 78 6 3 4 124

[0169] Monocytes 95 26 1 21 72

[0170] NK cells 104 17 2 11 81

[0171] B cells 104 10 0 16 85

[0172] CD4 T cells 116 8 2 16 73

[0173] CD8 T cells 109 9 4 15 78

[0174]

[0113] We did not observe many changes in modulatory receptor gene expression after activation in neutrophils. Six receptor genes, such as SLAMF1 , were absent on resting neutrophils, but expression was induced after stimulation, categorizing them as negative feedback receptors. We identified 124 receptors as threshold receptors, while 4 and 3 genes were identified as threshold-disinhibition and threshold-negative feedback receptors, respectively (Figure 2A).

[0175]

[0114] In contrast, in monocytes 26 genes, including SIGLEC8 or SIGLECH , had the expression pattern of negative feedback receptors. We found 72 receptors, e.g., SIGLEC10 or PILRA, that maintained their expression after activation, while 21 were downregulated, such as VSTM1 or CLEC12A. Only a single receptor, SLAMF7, was categorized as a threshold-negative feedback receptor. Of note, a cluster of 14 receptors was shared exclusively between monocytes and neutrophils (Figure 2B) and thus could be indicative of a set of conserved myeloid-specific checkpoints.

[0115] We observed expression of 17 negative feedback receptors, e.g., SIGLEC15 and VSTM4, and 81 threshold receptors, such as CD244 and CD300A on NK cells. Additionally, 11 genes were classified as threshold-disinhibition receptors, e.g., FCRL6, while 2 were determined to be threshold-negative feedback receptors.

[0176]

[0116] B cells expressed 111 predicted modulatory receptor genes, of which 10, like SIRPA and SIGLEC6, were negative feedback receptors. We found 85 threshold receptors, such as BTLA, as well as 16 threshold-disinhibition receptors, e.g., PILRA and LST1. B cells were the only subset that are not predicted to express thresholdnegative feedback receptors. B cells also shared expression of 6 receptors with monocytes and neutrophils, while 2 genes were uniquely expressed by B cells.

[0177]

[0117] CD4+ and CD8+ T cells expressed 8 and 9 negative feedback receptors, respectively, such as PDCD1 , in addition to 73 (on CD4+ T cells) and 78 (on CD8+ T cells) threshold receptors, e.g., CEACAM1 and SIT1. In addition, we found 16 threshold-disinhibition receptors on CD4+ T cells and 15 on CD8+ T cells, such as LAIR1 on CD4+ T cells and AXL on both subsets, as well as 2 threshold-negative feedback receptors on CD4+ and 4 on CD8+ T cells.

[0178]

[0118] All immune cell subsets expressed fewer multi-membrane spanning modulatory receptor genes compared to single-spanners. When we assigned these multi-spanning targets to the different functional categories, a similar picture emerged as for the single-spanners, where most receptors categorized as threshold receptors in all immune cell types. Whereas monocytes, B cells, CD4+ and CD8+ T cells expressed more single-spanning threshold-disinhibition receptors than negative feedback receptors, the opposite was observed for multi-spanning modulatory receptor genes. NK cells had less dynamic gene regulation of multi-spanning targets than of singlespanners, while categorization for neutrophils was similar between multi- and singlespanners (Figure 4A). Interestingly, the multi-spanning targets were shared between three or more subsets more often than was the case for the single-spanners (Figure 4B).

[0179]

[0119] In summary, we identified 398 known and putative modulatory receptors being expressed across a variety of immune cell subsets, with mostly overlapping expression patterns. We classified these receptors into different functional categories based on their expression in the resting and activated states. We found that all immune cells expressed mostly threshold receptors. Among the receptors that changed expression after activation, monocytes and NK cells mainly expressed negative feedback receptors, while the T cells and B cells expressed more threshold-disinhibition receptors. Neutrophils did not have very dynamic gene expression patterns.

[0180] Known and putative modulatory receptors are expressed on tumour infiltrating T cells

[0181]

[0120] To further translate our findings to a disease context, we explored the expression of putative modulatory receptor genes in tumour infiltrating lymphocytes. We used publicly available single cell RNA sequencing data of CD4+ and CD8+ T cells from 21 types of cancer and determined modulatory receptor gene expression in the different T cell subsets identified by Zheng et al. (2021)

[0021] (Figure 3A). After quality control and filtering, the expression of 133 / 215 single-spanners, and 111 / 197 of the multi-spanners could be assessed in this dataset.

[0182]

[0121] Among T cell subsets, expression of single-spanning modulatory receptor genes was variable (Data not shown). We found 27 and 46 modulatory receptor genes in naive CD4+ and CD8+ T cells, respectively, while T cells with a more differentiated phenotype, e.g., regulatory CD4+ T cells (68 genes) and exhausted CD8+ T cells (83 genes) expressed a wider array of known and predicted modulatory receptor genes (Figure 3A-B).

[0183]

[0122] When we assessed expression of multi-spanning modulatory receptor genes, we found similar variable expression across T cell subsets as for the single-spanners. Naive CD4+ T cells expressed 21 multi-spanning receptor genes, while many more were found in the other subsets (Figure 5A). Resident memory CD8+ T cells expressed 87 multi-spanning modulatory receptor genes, while CD8+ memory and effector memory expressed 9 and 26 genes, respectively (Figure 5B).

[0184]

[0123] Overall, this shows that the putative modulatory immune receptors we identified are expressed on tumor infiltrating T cells and differ between cellular subsets. Further investigation into differences and similarities between these populations in different cancer types could assist in targeting these receptors for therapy.

[0185]

[0124] In conclusion, we designed a novel, combined bioinformatics approach to predict around 400 immune modulatory receptors in the human genome, including both single and multi-spanning targets. Our list of novel targets will assist drug target selection in diseases where modulatory receptor targeting is warranted.

[0125] At the end of the Discussion, Tables 2a, 2b, 2c, 2d, 3a, 3a”, 3b, 3b” and 4 show the expressed modulatory immune receptors (HR) in the different settings disclosed in this Example.

[0186] DISCUSSION

[0187]

[0126] This study introduces some major improvements over previous attempts at predicting novel modulatory receptors in the human genome. In 2004, Staub et al., performed a search limited to type I membrane receptors containing an annotated extracellular domain and a restricted ITIM sequence. They identified a total of 94 genes encoding modulatory receptors, of which 32 had been previously described to rely on their ITIM to inhibit immune function

[0022] , A later study by Daeron et al. (2008), performed a more extensive analysis of ITIM-bearing receptors across multiple species, including chickens and Drosophila

[0012] , In humans, they identified 613 predicted type I and type II single-spanners and 992 multi-spanning transmembrane proteins, corresponding to around 1500 genes. This number agrees with what we retrieved in the initial steps of our bioinformatics pipeline, where we identified 1 ,562 genes. Our efforts to reduce this number to candidates with a high probability of functioning as modulatory receptors resulted in 390 novel targets. Compared to the previous studies, our method has key advantages. Firstly, the TOPCONS algorithm we used is superior to TMHMM and Philius, which have been used previously, at predicting membrane topology[14,23,24]. Most importantly, we excluded many potential false positives by filtering out proteins that are likely to contain an ITIM by chance and used three-dimensional structure predictions to enrich the remaining targets for those containing ITIMs in unstructured regions. Furthermore, our expression analysis and functional categorization of the putative modulatory receptors can assist in gaining a better understanding of their relevance in different contexts.

[0188]

[0127] An important component of our pipeline is the use of AlphaFold

[0017] , Our approach was exceptionally good at filtering out many proteins in the olfactory receptor family, for which almost all ITIMs identified were found in highly structured regions. We determined a threshold of 80 pLDDT based on the average prediction scores of the ITIM / ITSMs in known modulatory receptors. However, this excluded ITIMs in CD5 (Y402) and BTLA (Y282), with confidence scores of 85.2 and 81.5, respectively. Of note, studies have suggested that this particular ITIM in CD5 might not be functional or essential for CD5 function

[0025] . It is important to highlight that the AlphaFold structure predictions are based on the proteins being in isolation. It is possible that ITIMs become available after conformational changes following ligand binding, or that ITIMs in disordered regions are not functional when the proteins are found in complexes[26,27],

[0189]

[0128] We focused our search on receptors bearing ITIM or ITSM sequences, while modulatory receptors have been described that function independent of these short sequences. We previously reported that CD200R has a unique signaling motif that is not shared with any other protein

[0031] . Also, TIM-3 contains multiple tyrosine residues that are not part of any known signaling motif

[0032] , A small number (26 genes) of putative modulatory receptors contained an ITIM embedded in an ITAM sequence, i.e. , Yxx(l|L)x6-12Yxx(l|L). Although some ITAM-bearing molecules are capable of mediating modulatory signals in an ITIM-independent manner, these receptors may not all be modulatory

[0033] ,

[0190]

[0129] Until now, no known functional multimembrane spanning immune modulatory receptors have been reported, although some ITIM-bearing multi-spanners, e.g., SSTR2 and HCRTR1 , were shown to recruit SHP-2[34,35], It is possible that multispanning modulatory receptors have not yet been fully characterized due to their size and limited targetability, making them difficult to study. Of interest, in our analysis multi-spanning receptors were often expressed by multiple immune cell subsets. This may suggest that multi-spanning modulatory receptors would be capable of inhibiting a broad array of immune responses.

[0191]

[0130] We previously argued how different functional categories of modulatory receptors might serve distinct purposes[7]. Here, we grouped known and putative modulatory receptors in different functional categories based on their expression in isolated immune cell subsets in resting and stimulated state and show that a large number of them is expressed and regulated in a cell type-specific manner. However, as we categorized these receptors based on in vitro activation with limited stimuli, expression must be further validated in in vivo settings.

[0192]

[0131] We found that the highest number of target genes are expressed in neutrophils, which fits the need to control and precisely regulate their potentially damaging effector functions

[0036] . However, modulatory receptor gene expression on these cells did not change much after activation. This does not exclude regulation of receptor expression at the protein level, which would fit with the more short-lived nature of these cells. For instance, LAIR-1 protein is stored in intracellular granules which results in rapid membrane expression on activated granulocytes

[0036] . On monocytes, we found many negative feedback receptors. Considering that monocytes (especially macrophages) play a central role in orchestrating the overall immune response, timely resolution of their activation is key to successful resolution of inflammation, which could be achieved by these receptors.

[0193]

[0132] For the functional categorization of novel modulatory receptor genes, we collected multiple publicly available datasets containing different isolated immune cell subsets in the resting state and after cell type-specific activation. For 115 genes, we predicted a non-canonical isoform, but not the canonical sequence, as a putative modulatory receptor. This means that our expression analyses for these modulatory receptor genes might not be fully reflective of the expression of individual isoforms of those genes, considering that isoform usage can vary widely between different cells and activation states, as has been well described for CEACAM1 [37,38], In the present study, we also did not consider the ligand expression of these known and putative receptors. Regulation of modulatory receptors does not only occur at the level of the receptor itself, but the presence or absence of ligands, decoy receptors, and other interactions all add additional layers of regulation of control to the immune system[7],

[0194]

[0133] As proof-of-concept, we assessed expression of both known and novel targets in various tumor infiltrating T cell subsets. This helps understanding how these drug targets are differentially expressed in a disease context. As many receptors we identified were shared across immune cell subsets, it would be particularly interesting to block those that are enriched in, e.g., effector CD8+ T cell subsets. In contrast, receptors expressed in subsets that can have pro-tumorigenic properties, such as CD4+ Treg or Th17 cells[39,40], should not be activated; these targets could instead benefit from agonist therapy. Furthermore, these receptors are also attractive in autoimmune diseases, where immune checkpoints are increasingly being explored as targets for therapy[41 ,42],

[0195]

[0134] Taken together, we have designed a bioinformatic pipeline to identify a key list of 390 predicted modulatory receptors to be used as a starting point for further research into novel targets for targeting in diseases. Tables 2a, 2b, 2c, 2d, 3a, 3a”, 3b, 3b” and 4 show the expressed modulatory immune receptors (HR) in the different settings disclosed in this Example.

[0196]

[0197]

[0198]

[0199] MA TERIALS AND METHODS

[0200] Identification of putative modulatory receptors in the human genome

[0201]

[0135] To identify putative modulatory receptors in the human genome, amino acid sequences for 96,457 protein-coding, as annotated by Ensembl, transcripts were retrieved from Ensembl (release 105, December 2021)

[0013] using the biomaRt package

[0043] in R. Amino acid sequences were then filtered based on the presence of an ITIM or ITSM sequence, (V| L| I |S|T)xYxx(l | L| V). Seven out of 40,063 ITIM- containing proteins were larger than 10,000 amino acids, and were excluded because they are not feasible for membrane topology prediction using the TOPCONS method

[0014] . Topology results for the remaining 40,056 proteins were then retrieved, and proteins were further filtered based on the presence of a predicted transmembrane domain (12,576 out of 40,056 proteins). For the remaining proteins, we determined whether the identified ITIM or ITSM sequence corresponded to an intracellular section of the protein, which resulted in 4,613 protein sequences remaining, corresponding to 1 ,562 individual genes (Figure 1). A further 61 protein sequences were excluded as they could not be matched to a HUGO Gene Nomenclature Committee (HGNC) gene symbol

[0044] .

[0202] Determining the likelihood of intracellular protein domain containing an ITIM by chance

[0203]

[0136] To determine the odds of finding an intracellular ITIM or ITSM sequence in a specific protein, all intracellular domains of the proteins were retrieved based on the TOPCONS predictions. For each protein, the amino acids in the intracellular domain were then shuffled randomly, using the stringi package

[0045] , and the number of identified ITIM or ITSM sequences in the permutated sequences was compared to the actual number of intracellular ITIM or ITSM sequences in the original protein. Each protein was permutated 10,000 times, and the number of occurrences of a permutated protein containing more or equal ITIM / ITSMs to the original sequence was counted and used as measure for the likelihood of finding an ITIM or ITSM by chance. A threshold was defined based on likelihood scores for the proteins corresponding to known modulatory receptors, and was set at 0.25+E, with E being a random number determined error between 0 and 0.01.

[0204] Determining AlphaFold model confidence scores for individual ITIMs

[0205]

[0137] Three-dimensional structure of protein sequences that passed the likelihood threshold were next determined using AlphaFold (v2.2.0) under default parameters with amber minimization (run_relax = True) using the pdb70, uniref90, mgnify (mgy_clusters_2018_12), uniclust30 (uniclust30_2018_08) and bfd (bfd_metaclust_clu_complete_id30_c90_final_seq) databases on a high-performance cluster[17,46], AlphaFold was not able to determine the structure for 46 of these proteins, of which 6 were too long and 40 returned a run time error involving HHblits and the hh-suite. The three-dimensional prediction of 37 of these proteins was instead determined using ColabFold

[0047] (v1.5.2) using MMseqs2 and HHsearch under default parameters without using template information (template_mode = none) and using mmseq2_uniref_env as MSA mode. The top ranked prediction files were retrieved for each protein, and loaded into R using the bio3d package

[0048] . Prediction confidence scores (pLDDT) were retrieved, and the average pLDDT for all six amino acids encompassing the ITIM or ITSM sequence was used as the AlphaFold confidence score for that ITIM or ITSM. Proteins that had no ITIM or ITSM sequences with an average confidence score below 80 were used in further analysis.

[0206] Classification of receptor functional categories

[0207]

[0138] Based on the in vitro RNA sequencing dataset of immune cells in the resting state and after activation, functional categories were defined based on expression patterns as outlined previously[7]. Data was retrieved from Calderon et al. (2019) and West et al. (2016)[19,20] Gene symbols were matched to Ensembl Gene IDs using biomaRt and Ensembl (release 108, October 2022)

[0013] , For the neutrophil data

[0019] , TPM values were retrieved and Iog2 scaled. For other immune cells, count matrix was normalized using the DESeq2 package

[0049] . Culture conditions for all immune cells were simplified to major immune cell subsets, and to resting and stimulated (Table 2). Average expression for each modulatory receptor gene was determined for each sample. An modulatory receptor gene was considered expressed when it was above the median expression of all genes within the sample. Genes that were not expressed in the resting state, but were expressed in the stimulated state were considered negative feedback receptors. Genes expressed in the resting state were considered threshold, threshold-disinhibition, or threshold-negative feedback receptors if the Iog2 fold change in expression was between -0.5 and 0.5, below -0.5, or above 0.5, respectively. Genes expressed in the resting state, but not after activation were also considered threshold-disinhibition receptors.

[0208] Collection and normalization of immune cell RNAseq data

[0209]

[0139] For the analysis of tumour infiltrating T cells, a normalized expression matrix was retrieved from Zheng et al. (2021)[21 ,50], The average Z score of all cells in a T cell subset was determined, weighted by the relative contribution of different subclusters identified in the study. Modulatory receptor genes were considered expressed in a T cell subset if the average Z score was above 0.

[0210]

[0140] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

[0141] Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description, or embodiment of the present invention is disclosed, taught, or suggested in the relevant art.

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Claims

CLAIMS1. A method for screening for compounds which modulate the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell, more preferably wherein the ITIM and / or ITSM comprising modulatory immune receptor is i. a negative feedback modulatory immune receptor, preferably that is expressed in an activated immune cell; ii. a threshold modulatory immune receptor, preferably that is expressed on an immune cells in resting state; iii. a threshold-disinhibition modulatory immune receptor, preferably that is expressed on an immune cell in resting state and which expression is downregulated after activation of the immune cell; or iv. a threshold-negative feedback modulatory immune receptor, preferably expressed on an immune cell in resting state and which expression is upregulated after activation of the immune cell, comprising:(a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof with a test compound; and(b) determining the effect of the test compound on the expression and / or activity of the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof.

2. A method for identifying a compound which modulates the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell, more preferably wherein the ITIM and / or ITSM comprising modulatory immune receptor is i. a negative feedback modulatory immune receptor, preferably that is expressed in an activated immune cell;ii. a threshold modulatory immune receptor, preferably that is expressed on an immune cells in resting state; iii. a threshold-disinhibition modulatory immune receptor, preferably that is expressed on an immune cell in resting state and which expression is downregulated after activation of the immune cell; or iv. a threshold-negative feedback modulatory immune receptor, preferably expressed on an immune cell in resting state and which expression is upregulated after activation of the immune cell, comprising:(a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof with a test compound; and(b) determining the effect of the test compound on the expression and / or activity of the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof to thereby identify a compound which modulates the activity of the polypeptide or biologically active portion thereof.

3. A method for testing a drug for modulating the expression and / or activity of a ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell, more preferably wherein the ITIM and / or ITSM comprising modulatory immune receptor is i. a negative feedback modulatory immune receptor, preferably that is expressed in an activated immune cell; ii. a threshold modulatory immune receptor, preferably that is expressed on an immune cells in resting state; iii. a threshold-disinhibition modulatory immune receptor, preferably that is expressed on an immune cell in resting state and which expression is downregulated after activation of the immune cell; oriv. a threshold-negative feedback modulatory immune receptor, preferably expressed on an immune cell in resting state and which expression is upregulated after activation of the immune cell, comprising:(a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof with the drug; and(b) determining the effect of the drug on the expression and / or activity of the ITIM and / or ITSM comprising modulatory immune receptor or biologically active portion thereof.

4. The method of any of claims 1 - 3, wherein the ITIM and / or ITSM comprising modulatory immune receptor expressed in an immune cell is encoded by a gene listed in Table 1.

5. A method for screening for compounds which modulate the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising:(a) contacting the polypeptide or biologically active portion thereof with a test compound; and(b) determining the effect of the test compound on the expression and / or activity of the polypeptide or biologically active portion thereof.

6. A method for identifying a compound which modulates the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising:(a) contacting the polypeptide or biologically active portion thereof with a test compound; and(b) determining the effect of the test compound on the expression and / or activity of the polypeptide or biologically active portion thereof to thereby identify acompound which modulates the activity of the polypeptide or biologically active portion thereof.

7. A method for testing a drug for modulating the expression and / or activity of a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising:(a) contacting the polypeptide or biologically active portion thereof with the drug; and(b) determining the effect of the drug on the expression and / or activity of the polypeptide or biologically active portion thereof.

8. The method of any of claims 1 - 7, wherein the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide has the ability to modulate immunomodulatory function, modulate cytokine expression, modulate immune cell activation, modulate migration ability, modulate killing ability, and / or modulate cellular proliferation.

9. The method of any of claims 1 - 8, wherein the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide is expressed in a cell, preferably an immune cell.

10. The method of any of claim 1 - 9, wherein the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide is expressed in a cell, preferably an immune cells, and wherein the contacting with the test compound or drug is performed in the presence of a target of the cell, preferably immune cell.11 . The method of any of claims 1 - 10, wherein the immune cell is a neutrophil, a monocyte, a myeloid cell, a lymphoid cell, a B cell, a T cell, a NK cell, a helper T cell, a follicular helper T cell, an effector memory T cell, a regulatory T cell, an ‘unconventional’ T cell, a macrophage.

12. The method of any of claims 1 - 11 , wherein the T cell is a CD4+ T-cell, a CD8+ T cell, a tumor infiltrating T cell, an exhausted CD8+ T cell, a CD4+ memory T cell, an ‘unconventional’ T cell,.

13. The method of any of claim 1 - 12, wherein determining the effect of the test compound or the drug on activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide comprises determining an effect of the test compound or the drug on the immune response of the cell, preferably the immune cell expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide.

14. The method of claim 13, wherein the effect of the test compound or the drug on the immune response of the cell is modulating immunomodulatory function, modulating cytokine expression, modulating immune cell activation, modulating migration ability, modulating killing ability, and / or modulating cellular proliferation.

15. The method of any of claims 1 - 14, wherein the method comprises contacting the test compound or the drug with a cell expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide and contacting the test compound or the drug with an identical cell but not expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide or with an identical cell with reduced expression of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide or the polypeptide, and comparing the effect of the test compound or the drug on the expression and / or activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide.

16. The method of any of claims 1 - 15, wherein the cell is an immune cell and wherein the immune cell is activated prior to, during and / or subsequent to contacting the test compound or the drug with the cell.

17. The method of any of claim 1 - 16, wherein a further compound is contacted with the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptideprior to, during and / or subsequent to contacting the test compound or the drug with the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide, preferably wherein the further compound is a known modulatory immune receptor modulator.

18. The method of any of claims 1 - 17, wherein the cell is a cell obtained from a subject, preferably wherein the cell is obtained from a subject suffering from cancer or suffering from an immune disease or suffering from inflammation.

19. The method of any of claims 1 - 18, wherein the step of contacting occurs in vivo, ex vivo, or in vitro.

20. An immune cell wherein a gene encoding the ITIM and / or ITSM comprising modulatory immune receptor or a gene encoding the polypeptide encoded by a gene listed in Table 1 has been knocked-out.

21. An expression vector comprising a nucleotide sequence encoding the ITIM and / or ITSM comprising modulatory immune receptor or encoding the polypeptide encoded by a gene listed in Table 1 , operably linked with a promoter.

22. A host cell comprising the vector of claim 21 , or recombinantly expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1.

23. A method of determining the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 in a cell comprising:(a) providing the cell; and(b) determining the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 in the cell, preferably wherein the cell is an immune cell.

24. The method of claim 23 wherein the cell, prior to determining the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 , is contacted with a test compound or drug as defined in any of claims 1 - 7.

25. The method of any of claims 23 - 24, wherein the copy number, level of expression, or level of activity of the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide encoded by a gene listed in Table 1 is, preferably independently, determined in more than one cell type, preferably wherein the more than one cell type is obtained from a subject, preferably from a subject suffering from cancer or suffering from an immune disease or suffering from inflammation.

26. A method for identifying a binding partner to a ITIM and / or ITSM comprising modulatory immune receptor or a polypeptide encoded by a gene listed in Table 1 or biological active portion thereof comprising:(a) contacting the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide or biologically active portion thereof, or a cell expressing the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide, or biologically active portion thereof, with a test compound; and(b) determining whether the ITIM and / or ITSM comprising modulatory immune receptor or the polypeptide or biologically active portion thereof binds to the test compound.

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